Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Conjugate or complex
Reexamination Certificate
1999-03-09
2003-11-11
Devi, S. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Conjugate or complex
C424S194100, C424S193100, C424S184100, C424S203100, C424S234100, C424S250100, C514S023000, C514S054000, C536S123100
Reexamination Certificate
active
06645503
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to antigenic conjugates of the conserved portion of the lipopolysaccharides of certain gram negative bacteria and to vaccines containing such antigenic conjugates. The conjugates elicit antibodies which exhibit cross reactivity against heterologous strains of gram negative bacteria and the vaccines containing such conjugates induce antibodies which are functional and protective against such gram negative bacterial organisms.
BACKGROUND OF THE INVENTION
Lipopolysaccharides (LPS) are major surface antigens localized abundantly on the surface of gram negative bacteria. LPS molecules are comprised of: (1) a lipid A portion which consists of a glucosamine disaccharide substituted with phosphates, phosphoethanolamine groups and long chain fatty acids in ester and amide linkages; (2) an inner core portion attached to the lipid A portion by an eight carbon sugar, ketodeoxyoctonoate (KDO), which may be substituted by 1 to 2 additional KDO molecules and by up to 3 heptose moieties; (3) an outer core portion comprising hexoses such as glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine; and (4) an O-specific chain comprising repeating oligo-saccharide units which vary widely among bacterial strains. Polymerization of these repeating units to structures in excess of 60,000 daltons is not uncommon. The LPS molecules can vary extensively at the structural and antigenic level among bacterial strains, although the structure of the inner core is largely conserved among bacterial species. A typical structure of the lipid-A inner core of
Salmonella typhimurium
LPS is illustrated in FIG.
1
. The immune response responsible for the evolution of naturally protective antibodies is considered to arise by natural immunization to this region of the LPS.
In non-enteric pathogens, the LPS structure lacks repeating O-antigen units. Moreover, the complete genetic machinery for assembly of the O-antigen repeating unit appears to be absent in such pathogens. This has led to the designation of these LPS structures as lipooligosaccharides (LOS). There are similarities between LPS and LOS structures in such pathogens. For instance, all of the LPS and LOS structures link the lipid A regions to the cores through the KDO junction. The number of KDO residues can vary from one (e.g.,
Haemophilus influenzae
and
Haemophilus ducreyi
) to two (e.g.,
Neisseria meningitidis
and
Neisseria gonorrhoeae
). Recent studies indicate that the branched oliogsaccharides are synthesized separately from the core region and the assembly of the entire LOS structure is completed on the outer side of the cytoplasmic membrane (see Preston, et al., “The Lipooligosaccharides of Pathogenic Gram-Negative Bacteria”,
Critical Reviews In Microbiology
, 22:139-180 (1996)).
Accordingly, there is a single core region in such LOS structures without a distinct inner and outer core region. The core structure of these pathogens can vary from species to species and may comprise KDO in the complete absence of heptose (e.g.,
Moraxella catarrhalis
); KDO in the presence of a di-heptose structure (e.g.,
Neisseria meningitidis
and
Neisseria gonorrhoeae
); or KDO in the presence of a tri-heptose structure (e.g.,
Haemophilus influenzae
and
Haemophilus ducreyi
). Examples of core structures from Haemophilus and Neisseria are shown in FIG.
2
. The oligosaccharide units can extend from each of the heptoses and/or they can be substituted by phosphoethanolamine groups. Typical examples of completed LOS structures of
Haemophilus influenzae
strain 2019 (see Phillips et al., “Structural Characterization of the Cell Surface Lipooligosaccharides from a Non-Typable Strain of
Haemophilus Influenzae,” Biochemistry
, 31:4515-4526 (1992)) and
Neisseria gonorrhoeae
strain 1291 (see John et al., “The Structural Bases for Pyocin Resistance in
Neisseria gonorrhoeae
lipooligosaccharides,”
J. Biol. Chem
., 266:1903-1911 (1991)) are shown in FIG.
3
.
The use of LPS in the development of vaccines is known in the art. It has long been recognized that a specific antibody response directed against the LPS of a particular bacterial pathogen can contribute to protection against that specific strain. It is further known that saccharide structures (e.g., the saccharide portions of LPS) can be conjugated to a carrier protein, so that a vaccine composition containing such a conjugate will elicit the desired T-dependent response. An example of this is the successful glycoconjugate vaccines against bacteria having type-specific capsular saccharides see,
Vaccine Design: The Subunit and Adjuvant Approach
, Powell, M. F., and Newman, M. J., 673-694 (1995). This category of immune response is the basis for the effectiveness in human infants of a new generation of saccharide-protein conjugate vaccines as discussed in
Vaccine Design: The Subunit and Adjuvant Approach
, Powell, M. F., and Newman, M. J., 695-718 (1995).
However, since the LPS of heterologous strains of such pathogens demonstrate extensive variation of the outer core saccharide and/or O-specific chain, efforts to generate an antibody response to a number of heterologous strains or heterologous genera of bacteria utilizing a vaccine containing a single LPS have to date been unsuccessful.
In an effort to develop an LPS-based vaccine against
Neisseria meningitidis
, tetanus toxoid has been conjugated with oligosaccharides isolated from the LPS of a number of
Neisseria meningitidis
strains (see, Jennings et al.,
Infect. Immun
., 43:407-412 (1984)). However, the antibodies elicited by this conjugate were oligosaccharide specific and exhibited a high degree of serotype specificity.
Verheul et al.,
Infect. Immun
., 61:187-196 (1993), disclose the conjugation of oligosaccharides of meningococcal LPS to either tetanus toxoid or meningococcal outer membrane protein. In mice, the tetanus toxoid conjugates induced oligosaccharide specific antibodies which were not bactericidal against meningococci. The outer membrane protein—LPS conjugates induced antibodies against the outer membrane protein, but not against LPS. Verheul et al.,
Infect. Immun
., 59:843-851 (1991), also studied the immunogenicity of the conjugates of oligosaccharides of several
Neisseria meningitidis
strains and tetanus toxoid in rabbits. The results demonstrated that the antibodies elicited are directed only towards the oligosaccharide portion of the LPS which contain the immunotype specific epitopes.
The preparation of oligosaccharides from the LPS of
Neisseria meningitidis
laboratory adapted wild strain A1 and the subsequent conjugation thereof to tetanus toxoid as the carrier protein was disclosed in Gu et al.,
Infect. Immun
., 61:1873-1880 (1993). The conjugates were immunogenic in mice and rabbits and the majority of the antibodies were directed against immunotype-specific LPS epitopes. Also, the conjugate antisera showed less cross reactivity to different immunotypes of LPS than the LPS antisera. These studies demonstrate that LPS-derived oligosaccharide conjugates induce antibodies to the specific oligosaccharide immunotype. However, there was no evidence that the conjugate induced significant cross reactive antibodies to a common core saccharide structure present in the majority of
Neisseria meningitidis
strains.
Bhattacharjee et al.,
J. Infect. Dis
., 173:1157-1163 (1996) disclosed the mixture of the LPS from
Escherichia coli
with the outer membrane protein of
Neisseria meningitidis
group B, resulting in the formation of unconjugated, non-covalent complexes thereof. It was found that these complexes elicited antibodies which cross reacted with a number of gram negative bacteria. However, no evidence was provided to indicate that these complexes had properties different from other preparations of unconjugated saccharide structures which are known to be incapable of eliciting a T-dependent antibody response which can be boosted upon administration of subsequent doses. Moreover, it is known that such unconjugated saccharides do not elicit an appropriate immune respons
Apicella Michael A.
Arumugham Rasappa G.
Fortuna-Nevin Maria
Gibson Bradford W.
Devi S.
Nagy Michael R.
Szatkowski Thomas S.
Wyeth Holdings Corporation
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